The unifying goal of this proposal is to understand the structures and functions of machines that assemble on cellular membranes. Cells depend upon these machines in order to transform the shape, size and connectivity of their membranes and such remodeling underlies cell division, migration, differentiation, and communication. In addition, every pathogen hijacks or disrupts membrane-associated complexes to infect or escape from cells. Despite such central importance we lack comprehensive models of how membrane-binding proteins transduce signals, oligomerize, or remodel the size, shape and topology of cellular membranes. To overcome the intrinsic challenges in studying multi-component complexes that assemble transiently on membranes, we will develop genetic, biochemical and structural methods to discover and characterize bilayer-bound machines in molecular detail and to learn how they function within intricate cellular pathways. We build genome-scale maps of genetic interaction networks in model organisms to identify multi-component complexes and to infer their functions. We develop methods for reconstituting membrane-binding proteins in the presence of model membranes that mimic the in vivo target membrane in topology, lipid composition, size, and shape. We then leverage advances in cryo-electron microscopy (cryoEM) with our novel image analysis algorithms to solve structures of these machines in their native, membrane-associated states. These innovative tools are distinct methodologically but reinforcing: our genetic maps first define complexes for in depth in vitro study; and after reconstitute and solve 3D reconstructions by cryoEM we probe the functional predictions of our models with loss-of-function versus second-site suppression assays in vivo. These orthogonal approaches thus provide synergistic evidence for the mechanisms that drive cellular functions and result in genuine atomic-resolution understanding of the structures and functions we study. In concert with technology advances in electron optics and electron detectors, my laboratory is overcoming the genetic, biochemical and computational challenges responsible for the lack of structural models for membrane-bound protein assemblies. The aims outlined here are first, critical steps in our long-term research program to determine how the mechanisms we discover are corrupted by disease or hijacked by pathogens. Our ultimate objective is to translate this understanding into effective and tolerable treatments for diseases caused by defective or co-opted membrane-associated machines.